This invention relates to lineage-restricted intermediate precursor cells and methods of making thereof More particularly, the invention relates to neuronal-restricted precursors (NRP""s) isolated from mammalian embryos or mammalian neuroepithelial stem cells. These neuronal-restricted precursors are capable of self-renewal and differentiation into neurons, but not into glia, i.e. astrocytes and oligodendrocytes. Methods of generating, isolating, and culturing such neuronal-restricted precursor cells are also described.
Multipotent cells with the characteristics of stem cells have been identified in several regions of the central nervous system and at several developmental stages. F. H. Gage et al., Isolation, Characterization and Use of Stem Cells from the CNS, 18 Ann. Rev. Neurosci. 159-92 (1995); M. Marvin and R. McKay, Multipotential Stem Cells in the Vertebrate CNS, 3 Semin. Cell. Biol. 401-11 (1992); R. P. Skoff, The Lineages of Neuroglial Cells, 2 The Neuroscientist 335-44 (1996). These cells, often referred to as neuroepithelial stem cells (NEP cells), have the capacity to undergo self renewal and to differentiate into neurons, oligodendrocytes, and astrocytes, thus representing multipotent stem cells. A. A. Davis and S. Temple, A Self-Renewing Multipotential Stem Cell in Embryonic Rat Cerebral Cortex, 362 Nature 363-72 (1994); A. G. Gritti et al., Multipotential Stem Cells from the Adult Mouse Brain Proliferate and Self-Renew in Response to Basic Fibroblast Growth Factor, 16 J. Neurosci. 1091-1100 (1996); B. A. Reynolds et al., A Multipotent EGF-Responsive Striatal Embryonic Progenitor Cell Produces Neurons and Astrocytes, 12 J. Neurosci. 4565-74 (1992); B. A. Reynolds and S. Weiss, Clonal and Population Analyses Demonstrate that an EGF-Responsive Mammalian Embryonic CNS Precursor is a Stem Cell, 175 Developmental Biol. 1-13 (1996); B. P. Williams et al., The Generation of Neurons and Oligodendrocytes from a Common Precursor Cell, 7 Neuron 685-93 (1991).
The nervous system also contains precursor cells with restricted differentiation potentials. T. J. Kilpatrick and P. F. Bartlett, Cloned Multipotential Precursors from the Mouse Cerebrum Require FGF-2, Whereas Glial Restricted Precursors are Stimulated with Either FGF-2 or EGF, 15 J. Neurosci. 3653-61 (1995); J. Price et al., Lineage Analysis in the Vertebrate Nervous System by Retrovirus-Mediated Gene Transfer, 84 Developmental Biol. 156-60 (1987); B. A. Reynolds et al., supra; B. A. Reynolds and S. Weiss, supra; B. Williams, Precursor Cell Types in the Germinal Zone of the Cerebral Cortex, 17 BioEssays 391-93 (1995); B. P. Williams et al., supra. The relationship between multipotent stem cells and lineage restricted precursor cells is still unclear. In principal, lineage restricted cells could be derived from multipotent cells, but this is still a hypothetical possibility in the nervous system with no direct experimental evidence. Further, no method of purifying such precursors from multipotent cells has been described.
As has been shown in copending U.S. patent application Ser. No. 08/852/744, entitled xe2x80x9cGeneration, Characterization, and Isolation of Neuroepithelial Stem Cells and Lineage Restricted Intermediate Precursor,xe2x80x9d filed May 7, 1997, now U.S. Pat. No. 5,361,996, hereby incorporated by reference in its entirety, NEP cells grow on fibronectin and require fibroblast growth factor (FGF) and an as yet uncharacterized component present in chick embryo extract (CEE) to proliferate and maintain an undifferentiated phenotype in culture. The growth requirements of NEP cells are different from neurospheres isolated from E14.5 cortical ventricular zone cells. B. A. Reynolds et al., supra; B. A. Reynolds and S. Weiss, supra; WO 9615226; WO 9615224; WO 9609543; WO 9513364; WO 9416718; WO 9410292; WO 9409119. Neurospheres grow in suspension culture and do not require CEE or FGF, but are dependent on epidermal growth factor (EGF) for survival. FGF itself is not sufficient for long term growth of neurospheres, though FGF may support their growth transiently. NEP cells, however, grow in adherent culture, are FGF dependent, do not express detectable levels of EGF receptors, and are isolated at a stage of embryonic development prior to which it has been possible to isolate neurospheres. Thus, NEP cells may represent a multipotent precursor characteristic of the brain stem and spinal cord, while neurospheres may represent a stem cell more characteristic of the cortex. Nonetheless, NEP cells provide a model system for studying the principles of lineage restriction from multipotent stem cells or precursor cells of the central nervous system. The principles elucidated from the study of NEP cells are expected to be broadly applicable to all CNS precursor cells sufficiently multipotent to generate both neurons and glia. Thus, the present application is intended to be applicable to any CNS precursor cells regardless of their site of derivation as long as they are able to differentiate to both neurons and glial cells.
U.S. Pat. No. 5,589,376, to D. J. Anderson and D. L. Stemple, discloses mammalian neural crest stem cells and methods of isolation and clonal propagation thereof, but fails to disclose cultured NEP cells, cultured lineage restricted precursor cells, and methods of generating, isolating, and culturing thereof. Neural crest cells differentiate into neurons and glia of the peripheral nervous system (PNS), whereas the neuroepithelial stem cells differentiate into neurons and glia of the central nervous system (CNS).
The neuron-restricted precursor cells described herein are distinct from the NEP cells, neurospheres, and neural crest stem cells that have been described elsewhere. NEP cells are capable of differentiating into neurons or glia whereas NRP""s can differentiate into neurons, but not glia, and NEP cells and NRP""s display distinct cell markers. As mentioned above, neurospheres grow in suspension culture and do not require CEE or FGF, but are dependent on EGF for survival, whereas NRP cells grow in adherent culture and do not express detectable levels of EGF receptors. Further, neural crest cells differentiate into neurons and glia of the peripheral nervous system (PNS), whereas NRP cells differentiate into neurons of the central nervous system (CNS). NRP cells express polysialated or embryonic neural cell adhesion molecule (E-NCAM), but NEP cells, neurospheres, and neural crest cells do not. Therefore, NRP cells are different in their proliferative potential, expression of cell markers, and nutritional requirements from these other cell types.
The ability to isolate and grow mammalian neuronal-restricted precursor cells in vitro allows for of using pure populations of neurons for transplantation, discovery of genes specific to selected stages of development, generation of cell-specific antibodies for therapeutic and diagnostic uses such as for targeted gene therapy, and the like. Further, NRP cells can be used to generate subpopulations of neurons with specific properties, i.e. motoneurons and other neuronal cells for analyzing neurotransmitter functions and small molecules in high throughput assays. Moreover, the methods of obtaining NRP cells from NEP cells provides for a ready source of a large number of post-mitotic neurons. Post-mitotic cells obtained from a tumor cell line are already being commercially marketed (e.g., Clontech, Palo Alto, Calif.). The present invention is also necessary to understand how multipotent neuroepithelial stem cells become restricted to the various neuroepithelial derivatives. In particular, culture conditions that allow the growth and self-renewal of mammalian neuronal-restricted precursor cells are desirable so that the particulars of the development of these mammalian stem cells can be ascertained. This is desirable because a number of tumors of neuroepithelial derivatives exist in mammals, particularly humans. Knowledge of mammalian neuroepithelial stem cell development is therefore needed to understand these disorders in humans.
In view of the foregoing, it will be appreciated that isolated populations of mammalian lineage restricted neuronal precursor cells and methods of generating, isolating, and culturing such cells would be significant advancements in the art.
It is an object of the present invention to provide isolated (pure) populations of mammalian neuronal-restricted precursor cells and their progeny.
It is another object of the invention to provide methods of generating, isolating, culturing, and regenerating of mammalian lineage-restricted neuronal precursor cells and their progeny.
It is yet another object of the invention to provide a method for the generation of lineage-restricted neuronal precursor cells from and CNS multipotent precursor cell able to generate both neurons and glia.
It is a still further object of the invention to provide pure differentiated populations of neuronal cells derived from lineage-restricted neuronal precursor cells.
These and other objects can be achieved by providing an isolated, pure population of mammalian CNS neuron-restricted precursor cells. Preferably, such neuron-restricted precursor cells are capable of self-renewal, differentiation to CNS neuronal cells but not to CNS glial cells, and expressing embryonic neural cell adhesion molecule, but not expressing a ganglioside recognized by A2B5 antibody. These neuron-restricted precursor cells may or may not express nestin or xcex2-III tubulin. Thus, embryonic neural cell adhesion molecule is a defining antigen for these cells.
A method of isolating a pure population of mammalian CNS neuron-restricted precursor cells comprises the steps of:
(a) isolating a population of mammalian multipotent CNS stem cells capable of generating both neurons and glia;
(b) incubating the multipotent CNS stem cells in a medium configured for inducing the cells to begin differentiating;
(c) purifying from the differentiating cells a subpopulation of cells expressing a selected antigen defining neuron-restricted precursor cells; and
(d) incubating the purified subpopulation of cells in a medium configured for supporting adherent growth thereof.
In a preferred embodiment of this method, the selected antigen defining neuron-restricted precursor cells is embryonic neural cell adhesion molecule. The purification of the subpopulation of cells expressing the defining antigen can be by specific antibody capture, fluorescence activated cell sorting, magnetic bead capture, or any equivalent methods that isolate the cells expressing the defining antigen. Specific antibody capture, fluorescence activated cell sorting, and magnetic bead capture, as well as other equivalent methods, are well known in the art. Specific antibody capture is a preferred procedure for purifying such cells. In one preferred embodiment, the mammalian multipotent CNS stem cells are neuroepithelial stem cells.
Another method of isolating a pure population of mammalian CNS neuron-restricted precursor cells comprises the steps of:
(a) removing a sample of CNS tissue from a mammalian embryo at a stage of embryonic development after closure of the neural tube but prior to differentiation of glial and neuronal cells in the neural tube;
(b) dissociating cells comprising the sample of CNS tissue removed from the mammalian embryo;
(c) purifying from the dissociated cells a subpopulation expressing a selected antigen defining neuron-restricted precursor cells;
(d) plating the purified subpopulation of cells in feeder-cell-independent culture on a substratum and in a medium configured for supporting adherent growth of the neuron-restricted precursor cells; and
(e) incubating the plated cells at a temperature and in an atmosphere conducive to growth of the neuron-restricted precursor cells.
In a preferred embodiment of this method, the selected antigen defining neuron-restricted precursor cells is embryonic neural cell adhesion molecule. As described above, purification of the subpopulation of cells expressing a defining antigen can be by methods well known in the art, such as specific antibody capture, fluorescence activated cells sorting, and magnetic bead capture, and the like. Specific antibody capture is preferred.
A method of obtaining postmitotic neurons comprises:
(a) providing neuron-restricted precursor cells and culturing the neuron-restricted precursor cells in proliferating conditions; and
(b) changing the culture conditions of the neuron-restricted precursor cells from proliferating conditions to differentiating condition, thereby causing the neuron-restricted precursor cells to differentiate into postmitotic neurons. Changing the culture conditions can comprise adding retinoic acid to the basal medium, withdrawing a mitotic factor (such as fibroblast growth factor) from the basal medium, or adding a neuronal maturation factor (such as sonic hedgehog and brain-derived neurotrophic factor) to the basal medium.
Before the present neuronal-restricted precursor cells and methods of making thereof are disclosed and described, it is to be understood that this invention is not limited to the particular configurations, process steps, and materials disclosed herein as such configurations, process steps, and materials may vary somewhat. It is also to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof
It must be noted that, as used in this specification and the appended claims, the singular forms xe2x80x9ca,xe2x80x9d xe2x80x9can,xe2x80x9d and xe2x80x9cthexe2x80x9d include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to xe2x80x9can embryoxe2x80x9d includes reference to two or more embryos, reference to xe2x80x9ca mitogenxe2x80x9d includes reference to a mixture of two or more mitogens, and reference to xe2x80x9ca factorxe2x80x9d includes reference to a mixture of two or more factors.
In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.
As used herein, xe2x80x9cself renewalxe2x80x9d refers, for example, to the capability of a neuroepithelial stem cell to divide to produce two daughter cells, at least one of which is a multipotent neuroepithelial stem cell, or to the capability of a neuronal-restricted precursor cell to divide to produce two daughter cells, at least one of which is a neuronal-restricted precursor cell.
As used herein, xe2x80x9cclonal densityxe2x80x9d and similar terms mean a density sufficiently low enough to result in the isolation of single, non-impinging cells when plated in a selected culture dish. An illustrative example of such a clonal density is about 225 cells/100 mm culture dish.
As used herein, xe2x80x9cfeeder-cell-independent adherent culturexe2x80x9d and similar terms mean the growth of cells in vitro in the absence of a layer of different cells that generally are first plated on a culture dish to which the cells from the tissue of interest are then added. In feeder cell cultures, the feeder cells provide a substratum for the attachment of cells from the tissue of interest and additionally serve as a source of mitogens and survival factors. The feeder-cell-independent adherent cultures herein use a chemically defined substratum, for example fibronectin, and mitogens or survival factors are provided by supplementation of the liquid culture medium with either purified factors or crude extracts from other cells or tissues. Therefore, in feeder-cell-independent cultures, the cells in the culture dish are primarily cells derived from the tissue of interest and do not contain other cell types required to support the growth of cells derived from the tissue of interest.
As used herein, xe2x80x9ceffective amountxe2x80x9d means an amount of a growth factor or survival factor or other factor that is nontoxic but sufficient to provide the desired effect and performance. For example, an effective amount of FGF as used herein means an amount selected so as to support self renewal and proliferation of NEP cells when used in combination with other essential nutrients, factors, and the like.
The present invention is illustrated using neuron-restricted precursor cells isolated from the rat. The invention, however, encompasses all mammalian neuronal-restricted precursor cells and is not limited to neuronal-restricted precursor cells from the rat. Mammalian neuron-restricted precursor cells can be isolated from human and non-human primates, equines, canines, felines, bovines, porcines, ovines, lagomorphs, and the like.
Pluripotent stem cells in the central nervous system may generate differentiated neurons and glia either directly or through the generation of lineage-restricted intermediate precursors. In the developing retina, it appears that multipotent retinal precursors can generate any combination of differentiated cells even at their final division, indicating that intermediate precursors do not exist. In other regions of the central nervous system, in contrast, retroviral labeling studies have suggested the existence of lineage-restricted precursors that generate only one type of cell or a limited number of cell types. Intermediate stage precursors such as the bipotential oligodendrocyte-type-2-astrocyte precursor (O-2A) and a neuronal precursor have also been described in tissue culture studies. Yet, the generation of intermediate lineage-restricted precursors from pluripotent embryonic or adult stem cells or other stem cells capable of differentiating into neurons and glia has not been observed. Thus, the lineal relationship between pluripotent stem cells identified in culture and the committed precursors identified in vivo and in vitro has heretofore been unknown. Possible models of development have included (1) pluripotent and more committed stem cells representing lineally related cells or (2) such cells representing independent pathways of differentiation.
The developing spinal cord represents an ideal model for studying this differentiation. At embryonic day 10.5 (E10.5), the caudal neural tube appears as a homogeneous population of nestin-immunoreactive dividing cells in vivo and in vitro. These initially homogeneous cells are patterned over several days to generate neurons, oligodendrocytes, and astrocytes in a characteristic spatial and temporal profile. Neurogenesis occurs first on a ventro-dorsal gradient, with the earliest neurons becoming postmitotic on E13.5 in rats. Neurogenesis continues over an additional two days followed by differentiation of oligodendrocyte precursors and the subsequent differentiation of astrocytes.
Methods for growing neuroepithelial stem (NEP) cells isolated from E10.5 rat embryos as undifferentiated cells for extended periods in vitro have been described in Ser. No. 08/852,744, and it has been shown further that these populations were able to generate the three major cell types in the CNS. Thus, NEP cells represent a dividing multipotent stem cell that may differentiate into neurons either via an intermediate neuroblast or directly as a part of its terminal differentiation. To determine whether neurons differentiated from NEP cells via intermediate, more-restricted precursors, a variety of immunologically defined populations from differentiating cultures of NEP cells were isolated and characterized. It is shown herein that cells morphologically and phenotypically identical to NRP""s can be isolated from NEP cell cultures. Clonal analysis shows that individual NEP cells generate neurons via the generation of neuronal precursors and that individual NEP cells can generate neuron-restricted and glial-restricted precursors. It is further shown that E-NCAM+(embryonic neural cell adhesion molecule positive) cells are present in E13.5 neural tube cultures and that these cells are mitotic, self renewing stem cells that can generate multiple neuronal phenotypes, but not astrocytes or oligodendrocytes. Thus, neuron restricted precursors (NRP""s) are an identifiable stage in the in vivo differentiation of neurons. These data provide a demonstration of a direct lineal relationship between multipotent and neuron-restricted stem cells and suggest that neural differentiation involves progressive restriction in developmental fate.
FIG. 1 presents a model for spinal cord differentiation. This model is similar to that proposed for hematopoiesis and for differentiation of neural crest (see review by D. J. Anderson, The Neural Crest Lineage Problem: Neuropoiesis?, 3 Neuron 1-12 (1989)). According to this model, NEP cells 10 represent a homogeneous population of cells in the caudal neural tube that express nestin (i.e. nestin+) but no other lineage marker (linxe2x88x92). These cells divide and self renew in culture and generate differentiated phenotypes. Previous data have suggested intermediate dividing precursors with a more restricted potential. R. H. Miller and V. Szigeti, infra; B. C. Warf et al., supra; N. P. Pringle and W. D. Richardson, supra; J. Ray and F. Gage, Spinal Cord Neuroblasts Proliferate in Response to Basic Fibroblast Growth Factor, 14 J. Neurosci. 3548-64 (1994). Such precursors include those precursors 14 that generate oligodendrocytes and type 2 astrocytes, bipotent astrocyte and neuronal precursors (not shown in FIG. 1), as well as neuronal progenitors 18 that generate several kinds of neurons 22, 26, 30. The model therefore suggests that the multipotent precursors (NEP cells) generate differentiated cells (i.e., oligodendrocytes, type 2 astrocytes, type 1 astrocytes, neurons, and motoneurons) through intermediate precursors. Consistent with this model are the results presented herein showing the existence of cells with a neuron-restricted proliferative potential.
NEP cell cultures provide a large source of transient cells that can be sorted to obtain differentiated cell types. The results described herein provide direct evidence to support a model describing initially multipotent cells undergoing progressive restriction in developmental potential under extrinsic influence to generate the different phenotypes within the CNS. Evidence is provided that initially multipotent NEP cells generate neuron-restricted precursors in vitro and that such neuron-restricted precursors are also present in vivo. It is also shown that NRP""s fulfill criteria of blast cells and that a direct lineal relationship between multipotent stem cells and more restricted NEP cells exists.
The results presented herein support that E-NCAM-immunoreactive cells are restricted in their developmental potential. E-NCAM+ cells fail to differentiate into oligodendrocytes or astrocytes under any culture conditions tested. In contrast, NEP cells differentiate into neurons, astrocytes, and oligodendrocytes, and A2B5-immunoreactive cells differentiate into oligodendrocytes under identical conditions. For these reasons, E-NCAM-immunoreactive cells are described herein as neuron-restricted precursors or NRP""s.
Immunopanning and double-labeling data demonstrate that E-NCAM can be used to identify a specific neuronal sublineage that is generated from multipotential NEP cells. Like markers for intermediate precursors in the hematopoietic system and neural crest, however, E-NCAM, and the A2B5 glial precursor marker as well, is not unique to intermediate precursors. E-NCAM has been shown to label some astrocytes. Similarly, A2B5 has been shown to recognize neurons in some species and is transiently expressed by astrocytes in some culture conditions. Nevertheless, under the specific culture conditions defined herein these markers can be used to select intermediate precursors and therefore represent the first cell surface epitopes that are co-expressed in concordance with a restriction in developmental potential.
The basal medium (NEP medium) used in the experiments described herein comprises DMEM-F12 (GIBCO/BRL, Gaithersburg, Md.) supplemented with 100 xcexcg/ml transferrin (Calbiochem, San Diego, Calif.), 5 xcexcg/ml insulin (Sigma Chemical Co., St. Louis, Mo.), 16 gig/ml putrescine (Sigma), 20 nM progesterone (Sigma), 30 nM selenious acid (Sigma), 1 mg/ml bovine serum albumin (GIBCO/BRL), plus B27 additives (GIBCO/BRL), 20 ng/ml basic fibroblast growth factor (bFGF), and 10% chick embryo extract (CEE). In general, these additives were stored as 100X concentrates at xe2x88x9220xc2x0 C. until use. Normally, 200 ml of NEP medium was prepared with all additives except CEE and used within two weeks of preparation. CEE was added to the NEP medium at the time of feeding cultured cells.
FGF and CEE were prepared as described in D. L. Stemple and D. J. Anderson, supra; M. S. Rao and D. J. Anderson, supra; L. Sommers et al., Cellular Function of the bHLH Transcription Factor MASH1 in Mammalian Neurogenesis, 15 Neuron 1245-58 (1995), hereby incorporated by reference. FGF is also available commercially (UBI).
Briefly, CEE was prepared as follows. Chick eggs were incubated for 11 days at 38xc2x0 C. in a humidified atmosphere. Eggs were washed and the embryos were removed and placed in a petri dish containing sterile Minimal Essential Medium (MEM with glutamine and Earle""s salts) (GIBCO/BRL) at 4xc2x0 C. Approximately 10 embryos each were macerated by passage through a 30-ml syringe into a 50-ml test tube. This procedure typically produced about 25 ml of medium. To each 25 ml was added 25 ml of MEM. The tubes were rocked at 4xc2x0 C. for 1 hour. Sterile hyaluronidase (1 mg/25 g of embryo) (Sigma) was added, and the mixture was centrifuged for 6 hours at 30,000 g. The supernate was collected, passed through a 0.45 xcexcm filter and then through a 0.22 xcexcm filter, and stored at xe2x88x9280xc2x0 C. until use.
Laminin (Biomedical Technologies Inc.) was dissolved in distilled water to a concentration of 20 mg/ml and applied to tissue culture plates (Falcon). Fibronectin (Sigma) was resuspended to a stock concentration of 10 mg/ml and stored at xe2x88x9280xc2x0 C. and then diluted to a concentration of 250 xcexcg/ml in D-PBS (GIBCO/BRL). The fibronectin solution was applied to tissue culture dishes and immediately withdrawn. Subsequently, the laminin solution was applied and plates were incubated for 5 hours. Excess laminin was withdrawn, and the plates were allowed to air dry. Coated plates were then rinsed with water and allowed to dry again. Fibronectin was chosen as a growth substrate for NEP cells because NEP cells did not adhere to collagen or poly-L-lysine (PLL) and adhered poorly to laminin. Thus, all subsequent experiments to maintain NEP cells in culture were performed on fibronectin-coated dishes. Laminin-coated dishes were used, however, to promote differentiation of NEP stem cells.
For clonal analysis, cells harvested by trypsinization were plated at a density of 50-100 cells per 35 mm dish. Individual cells were identified and located on the dish by marking the position with a grease pencil. Cells were grown in DMEM/F12 with additives, as described above, for a period ranging from 10-15 days.